1. Field of the Invention
The present invention relates to a polymer-siRNA delivery carrier in which a siRNA is combined with a polymer and the use thereof. More specifically, the present invention relates to stable polymer-siRNA nanoparticles in which a polymer and a siRNA are combined by using charge interaction and biodegradable covalent bonding at the same time and the use thereof.
2. Background Art
In recent years, the core technology of gene therapy depends on how oligonucleotide having strong negative charge is delivered into a desired tissue. In general, live cells have a very low permeability to high molecular weight molecules such as proteins or oligonucleotides. Since only some low molecular weight materials can enter the cytoplasm or nucleus within cells through the membrane of live cells at a very low rate, thereby causing a limitation when delivering high molecular weight molecules containing proteins or oligonucleotides. In order to overcome such a limitation, studies on various gene delivery carriers and their delivery method have been carried out. One of them is a delivery method using various viruses. The delivery method using viruses is excellent in the aspect of delivery efficiency but has a problem in applying to clinics due to an effect on the gene expression function of a host by virus genes and its carcinogenic possibility. Accordingly, it is required the development of delivery carriers for allowing stable gene delivery in the body while maintaining a high transfection efficiency of viruses.
RNA interference (RNAi) is primarily used in the gene therapy field as a treatment showing a prominent effect in reducing the expression of a specific gene. Due to its high activity and precise gene selectivity, siRNA is expected to be an alternative treatment to antisense oligonuceotide (ODN) currently being used as a therapeutic agent as a result of the past 20-year's research. Accordingly, more than thirty pharmaceutical and bio technology companies have concentrated on the development of a treatment drug based on siRNA. Particularly, the development of siRNA-related treatments for treating diabetes, obesity, rheumatism, Parkinson's disease, B/C-type hepatitis, AIDS, and cancer is in progress.
siRNA, which is a short, double-stranded RNA consisting of about 19 to 23 nucleotides, suppresses the expression of a gene by targeting the mRNA of a target gene having a complementary base sequence to them. In other words, an mRNA regulating the expression metabolic process of a specific gene is singularly degraded to stop the protein synthesis of the target gene, thereby treating a disease. Accordingly, studies on delivery carriers using cationic liposomes or micelles and cationic polymers for delivering siRNA having strong negative charge into a living body have been carried out. siRNA has a low stability and thus it is degraded in a short period of time by a variety of enzymes existing in plasma in large quantities in vivo. Particularly, in case of an injection treatment, it is more quickly destroyed if not stably treated chemically, and difficult to easily permeate membranes having negative charge due to its cationic property, and as a result, the transmissivity into cells is reduced, thereby causing a problem that the treatment efficiency is rapidly reduced. Although siRNA consists of double strands, the binding of ribose sugars constituting a RNA is very unstable chemically compared to the binding of deoxyribose sugars constituting a DNA, and thus most of them are rapidly degraded in vivo with a half-life of around 30 minutes. Furthermore, siRNA is recognized as foreign substances in vivo, thereby causing adverse effects on an immune system. Moreover, siRNA affects other portions of the gene that is not an originally planned portion of the gene, thereby causing cross-hybridization in a gene base sequence.
Accordingly, it is important to develop delivery carriers capable of neutralizing negative charge to allow in vivo permeation while overcoming the shortcoming of such siRNA in which treatment efficiency is rapidly reduced due to its low stability that is easily degraded in vivo. Preferably, the siRNA delivery carrier as a treatment has a singular accumulation in the internal circulation and specific disease portion, and should be suitably bio-degraded in the body. Glycol chitosan, which is one of bio-derived polymers, has been already known for the superiority as an anti-cancer delivery carrier due to its biocompatibility and biodegradability, and excellent cancer singular accumulation. Our laboratory has developed a siRNA delivery carrier (glycol chitosan-PEI) supplemented with positive charge by adding polyethyleneimine (PEI) strongly coupled with nucleotide to glycol chitosan (Korean Patent No. 2009-0041428). However, the cohesion is insufficient with the charge of a polymer alone in forming an effective composite with siRNA having strong negative charge.
On the other hand, there have been efforts for increasing the molecular weight of siRNA itself to complement the weak in vivo stability of low molecular weight siRNA, thereby allowing effective coupling with polymer delivery carriers. The Jean-Paul Behr group has reported that in vivo delivery efficiency of siRNA is increased by using sticky siRNA produced by adding DNA sequence to the 3′ end of siRNA (Proceedings of the National Academy of Sciences of the United States of America, 2007. 104 (41): 16050-16055), and our laboratory has released as a patent and a paper that in vivo stability and gene expression effect are increased by producing poly-siRNA in which a thiol group is introduced at the 5′ end of siRNA to increase the size by a disulfide bond between siRNAs (Korean Patent No. 10-2009-0042273, Journal of Controlled release, 2010. 141 (3): 339-346). Besides, for a method of producing poly-siRNA in which the size is increased to secure stability, there has been reported that the expression of a target gene is suppressed if a disulfide bond is introduced as a cross-linker to produce multimeric siRNA (Nature materials, 2010. 9: 272-278).
Hence, the present inventor has produced a polymer-siRNA delivery carrier in which a polymer having excellent biocompatibility and a siRNA are connected by charge interaction as well as biodegradable covalent bonding to increase the in vivo stability and delivery efficiency to a target portion of the siRNA that can be used as an effective gene treatment. Specifically, it has been developed a polymer-siRNA delivery carrier, which is a novel nano delivery carrier applicable to various disease treatments, by connecting a siRNA, poly-siRNA or multimeric siRNA to a polymer having suitable positive charge and containing a functional group using charge interaction as well as covalent bonding to a functional group at the end of siRNA, thereby blocking the expression of a specific protein of disease cells.